The genome organization of the recently sequenced 3.6 Mb human Major Histocompatibility Complex (MHC) will be best understood in a comparative evolutionary context. To develop a physical map of the domestic cat major histocompatibility complex (MHC) for nucleotide sequencing, we have constructed two large insert domestic cat genomic DNA libraries, one using the P1 artificial chromosome (PAC) system with an average insert size of 80 kb and a second using the bacterial artificial chromosome (BAC) system with an average insert size of 137 kb which has been arrayed in 384-well plates. State-of-the-art, high-throughput analysis technologies are being applied to BAC/PAC contig mapping of the feline MHC. Using feline and/or human MHC DNA probes, we have thus far identified 452 BAC and 66 PAC DNA clones containing the feline MHC. Putative MHC positive BAC/PAC clones are arranged in a 96-well plate format and rescreened by colony hybridization and BAC/PAC DNAs are purified for fingerprint analysis and Southern hybridization. To generate contig maps of large insert clones, complete Hind III digested BAC/PAC DNAs are fingerprinted by electrophoresis through agarose, stained, imaged, and the data analyzed using the Fingerprinting Contigs (FPC) software developed by C. Soderlund which assembles a minimal tiling path of clones using an algorithm to cluster clones into contigs based on their probability of coincidence scores. Using these appoaches, we have constructed a sequence ready 660 kbp BAC/PAC contig map of the domestic cat MHC class II region consisting of 24 BACs and 12 PACs with a gene order similar to, but distinct from human and mice: DPB/DPA, Ring3, DMB, TAP1, DOB, DRB2, DRA3, DRB2, DRA2 and DRA1. FISH analyses of selected class I and II PAC clones confirmed that the domestic cat MHC lies in the pericentromeric region of chromosome B2. However, apparently unlike the human and mouse MHC, both the domestic cat DRA and DRB genes have undergone multiple duplications, functional DPB/DPA genes have been deleted as well as the entire DQB/DQA region.